The present invention provides methods of use and pharmaceutical compositions for related genera of low molecular weight compounds comprising optionally substituted iminoquinone and orthoquinoid ring systems. The compounds have MIF (macrophage migration inhibitory factor) antagonist activity and find utility as such. For example, the MIF antagonists are useful in methods for treating a variety of diseases involving inflammatory activity or pro-inflammatory cytokine responses, such as autoimmune diseases, asthma, arthritis, EAE, ARDS (acute respiratory distress syndrome) and various forms of sepsis and septic shock, and other conditions characterized by underlying MIF responses including, for instance, tumor growth and neovascularization.
Macrophage migration inhibitory factor (MIF) is one of the earliest described cytokines, and is an immunoregulatory protein with a wide variety of cellular and biological activities (for reviews see: Swope and Lolis, Rev. Physiol. Biochem. Pharmacol. 139:1-32, 1999; Metz and Bucala, Adv. Immunol. 66:197-223, 1997; and Bucala, FASEB J. 14:1607-1613, 1996). Originally, MIF was found to be secreted by activated lymphoid cells, to inhibit the random migration of macrophages, and to be associated with delayed-type hypersensitivity reactions (George and Vaughan, Proc. Soc. Exp. Biol. Med. 111:514-521, 1962; Weiser et al., J. Immunol. 126:1958-1962, 1981; Bloom and Bennett, Science, 153:80-82, 1966; David, Proc. Natl. Acad. Sci. USA 56:72-77, 1966). MIF was also shown to enhance macrophage adherence, phagocytosis and tumoricidal activity (Nathan et al., J. Exp. Med. 137:275-288, 1973; Nathan et al., J. Exp. Med. 133:1356-1376, 1971; Churchill et al., J. Immunol. 115:781-785, 1975). Unfortunately, many of these early studies used mixed-culture supernatants that were shown later to contain other cytokines, such as IFN-xcex3 and IL-4 that also have macrophage migration inhibitory activity (McInnes and Rennick, J. Exp. Med. 167:598-611, 1988; Thurman et al., J. Immunol. 134:305-309, 1985) making the historical attribution of specific biological activities to the single protein now defined as MIF somewhat problematic. The availability of recombinant MIF has allowed for confirmation of these biological activities, and for the identification of additional activities.
Recombinant human MIF was originally cloned from a human T cell library (Weiser et al., Proc. Natl. Acad. Sci. USA 86: 7522-7526, 1989), and was shown to activate blood-derived macrophages to kill intracellular parasites and tumor cells in vitro, to stimulate IL-1xcex3 and TNFxcex1 expression, and to induce nitric oxide synthesis (Weiser et al., J. Immunol. 147:2006-2011, 1991; Pozzi et al., Cellular Immunol. 145:372-379, 1992; Weiser et al., Proc. Natl. Acad. Sci. USA 89:8049-8052, 1992; Cunha et al., J. Immunol. 150:1908-1912, 1993). While the conclusions available from several of these early reports are confounded by the presence of a bioactive mitogenic contaminant in the recombinant MIF preparations used, the potent pro-inflammatory activities of MIF have been re-defined in other studies that do not suffer from this complicating factor (reviewed in Bucala, The FASEB Journal 10:1607-1613, 1996).
More recent MIF studies have capitalized on the production of recombinant MIF in purified form as well as the development of MIF-specific polyclonal and monoclonal antibodies to establish the biological role of MIF in a variety of normal homeostatic and pathophysiological settings (reviewed in Rice et al., Annual Reports in Medicinal Chemistry 33:243-252, 1998). Among the most important insights of these later reports has been the recognition that MIF not only is a cytokine product of the immune system, but also is a hormone-like product of the endocrine system, particularly the pituitary gland. This work has underscored the potent activity of MIF as a counter-regulator of the anti-inflammatory effects of the glucocorticoids (both those endogenously released and those therapeutically administered), with the effect that the normal activities of glucocorticoids to limit and suppress the severity of inflammatory responses are inhibited by MIF. The endogenous MIF response is thus seen as a cause or an exacerbative factor in a variety of inflammatory diseases and conditions (reviewed in Donnelly and Bucala, Molecular Medicine Today 3:502-507, 1997).
MIF is now known to have several biological functions beyond its long-hypothesized association with delayed-type hypersensitivity reactions. For example, as mentioned above, MIF released by macrophages and T cells acts as a pituitary mediator in response to physiological concentrations of glucocorticoids (Bucala, FASEB J. 14:1607-1613, 1996). This leads to an overriding effect of glucocoticoid immunosuppressive activity through alterations in TNF-xcex1, IL-1xcex2, IL-6, and IL-8 levels. Additional biological activities of MIF include the regulation of stimulated T cells (Bacher et al., Proc. Natl. Acad. Sci. USA 93:7849-7854, 1996), the control of IgE synthesis (Mikayama et al., Proc. Natl. Acad. Sci. USA 90:10056-60, 1993), the functional inactivation of the p53 tumor suppressor protein (Hudson et al., J. Exp. Med. 190:1375-1382, 1999), the regulation of glucose and carbohydrate metabolism (Sakaue et al., Mol. Med. 5:361-371, 1999), and the attenuation of tumor cell growth and tumor angiogenesis (Chesney et al., Mol Med. 5:181-191, 1999; Shimizu et al., Biochem. Biophys. Res. Commun. 264:751-758, 1999).
MIF shares significant sequence homology (36% identity) with D-dopachrome tautomerase, and MIF has enzymatic activity to catalyze the tautomerization of the non-physiological substrates D-dopachrome (Rosengren et al., Mol. Med. 2:143-149, 1996) and L-dopachrome methyl ester (Bendrat et al., Biochemistry, 36:15356-15362, 1997) (FIG. 1A). Additionally, phenylpyruvic acid and p-hydroxyphenylpyruvic acid (Rosengren et al., FEBS Letter, 417:85-88, 1997), and 3,4-dihydroxyphenylaminechrome and norepinephrinechrome (Matsunaga et al., J. Biol. Chem., 274:3268-3271, 1999) are MIF substrates, although it is not known if tautomerization of any of these agents comprises a natural function for MIF.
The three-dimensional crystal structure of human MIF reveals that the protein exists as a homotrimer (Lolis et al., Proc. Ass. Am. Phys. 108:415-419, 1996) and is structurally related to 4-oxalocrotonate tautomerase, 5-(carboxymethyl)-2-hydroxymuconate isomerase, chorismate mutase, and to D-dopachrome tautomerase (Swope et al., EMBO J. 17:3534-3541, 1998; Sugimoto et al., Biochemistry, 38:3268-3279, 1999). Recently, the crystal structure has been reported for the complex formed between human MIF and p-hydroxyphenylpyruvic acid (Lubetsky et al., Biochemistry, 38:7346-54, 1999). It was found that the substrate binds to a hydrophobic cavity at the amino terminus and interacts with Pro-1, Lys-32, and Ile-64 in one of the subunits, and with Tyr-95 and Asn-97 in an adjacent subunit. Similar interactions between murine MIF and (E)-2-fluoro-p-hydroxycinnamate have been reported (Taylor et al., Biochemistry, 38:7444-7452, 1999). Solution studies using NMR provide further evidence of the interaction between p-hydroxyphenylpyruvic acid and Pro-1 in the amino-terminal hydrophobic cavity (Swope et al., EMBO J., 17:3534-3541, 1998).
Mutation studies provide convincing evidence that Pro-I is involved in the catalytic function of MIF. Deletion of Pro-1 or replacement of Pro-1 with Ser (Bendrat et al., Biochemistry, 36:15356-15362, 1997), Gly (Swope et al., EMBO J., 17:3534-3541, 1998), or Phe (Hermanowski-Vosatka et al., Biochemistry, 38:12841-12849, 1999), and addition of an N-terminal peptide tag to Pro-1 (Bendrat et al., Biochemistry, 36:15356-15362, 1997) abrogated the catalytic activity of MIF in assays using L-dopachrome methyl ester and p-hydroxyphenylpyruvic acid. A similar loss in activity was found by inserting Ala between Pro-1 and Met-2 (Lubetsky et al., Biochemistry, 38:7346-54, 1999), and by derivatization of Pro-1 with 3-bromopyruvate (Stamps et al., Biochemistry 37:10195-10202, 1998).
The connection between the enzyme and biological activities, however, remains unclear. The Pro to Ser MIF mutant showed glucocorticoid counter-regulatory activity (Bendrat et al., Biochemistry, 36:15356-15362, 1997) and was fully capable, as was the Pro to Phe mutant, of inhibiting monocyte chemotaxis (Hermanowski-Vosatka et al., Biochemistry, 38:12841-12849, 1999). In contrast, the Pro to Gly MIF mutant was greatly impaired in its activity to stimulate superoxide generation in activated neutrophils (Swope et al., EMBO J., 17:3534-3541, 1998). These results suggest that specific biological activities of enzymatically inactive MIF mutants may be differentially sensitive to specific mutations, reflected in differential effects in specific assays that are used to assess biological function.
There is a need in the art to discover and develop small organic molecules that function as MIF antagonists and further posses the benefits of small organic molecule therapeutics versus larger, oligomeric protein-(e.g., antibody) and nucleic acid-based (e.g., anti-sense) therapeutic agents. The therapeutic potential of low molecular weight MIF inhibitors is substantial, given the activities of anti-MIF antibodies in models of endotoxin- and exotoxin-induced toxic shock (Bernhagen et al., Nature, 365:756-759, 1993; Kobayashi et al., Hepatology, 29:1752-1759, 1999; Calandra et al., Proc. Natl. Acad. Sci. USA., 95:11383-11388, 1998; and Makita et al., Am. J. Respir. Crit. Care Med. 158:573-579, 1998), T-cell activation (Bacher et al., Proc. Natl. Acad. Sci. USA. 93:7849-7854, 1996), autoimmune diseases (e.g., graft versus host disease, insulin-dependent diabetes, and various forms of lupus) including rheumatoid arthritis (Kitaichi et al., Curr. Eye Res., 20:109-114, 2000; Leech et al., Arthritis Rheum., 42:1601-1608, 1999), wound healing(Abe et al., Biochim. Biophys. Acta, 1500:1-9, 2000), and angiogenesis (Shimizu et al., Biochem. Biophys. Res. Commun. 264:751-758, 1999). Low molecular weight anti-MIF antagonists (drugs) may offer clinical advantages over neutralizing antibodies and nucleic acid-based agents because the drug-like therapeutics may be orally active or generally more easily administered, have better bioavailabilities, have improved biodistributions, and should be much cheaper to produce. Prior to the present invention, the only published report of potent low molecular weight MIF inhibitors concerned some commonly found long chain fatty acids that reversibly inhibited the dopachrome tautomerase activity of mouse MIF (Bendrat et al., Biochemistry, 36:15356-15362, 1997). These fatty acids were never tested for their effects in biological assays of MIF activity.
The enzyme activity (tautomerase) of MIF and the substrates it accepts provide an enzymatic activity assay for designing low molecular weight agents that bind to MIF and disrupt its biological activity. The present invention provides methods of use for two related genera of such compounds having either iminoquinone-related or orthoquinoid-type structures. The iminoquinone-derived compounds are related to acetaminophen and some of its active metabolites. These agents react covalently with MIF, block its enzymatic activity, and have effects on MIF biological activity.
The present invention provides a method for treating inflammatory disorders including, but not limited to, arthritis, proliferative vascular disease, EAE, ARDS (acute respiratory distress syndrome), cytokine-mediated toxicity, sepsis, septic shock, psoriasis, interleukin-2 toxicity, asthma, MIF-mediated conditions, autoimmune disorders (including but not limited to, rheumatoid arthritis, insulin-dependent diabetes, multiple sclerosis, graft versus host disease, lupus syndromes), tumor growth or angiogenesis, or any condition characterized by local or systemic MIF release or synthesis, comprising administering an effective amount of a compound of formula I or formula II wherein formula I is: 
wherein R1, R2, R3, and R4 are independently H, CH3, CH2CH3, OH, OCH3, OCH2CH3, or halo (Br, Cl, F, or I) and R5 is independently H, CH3, CH2CH3, OH, OCH3, OCH2CH3, or NR6R7 wherein R6 and R7 are independently H or C1-4alkyl; and wherein formula II is: 
wherein R1, R2 and R3 are independently as defined above and R8 is (CHxe2x95x90CH)nxe2x80x94COxe2x80x94R5 wherein n=0, 1, 2, or 3 and R5 is independently as defined above for formula I.
Preferably for formula I: R5 is CH3; and R1, R2, R3 and R4 are independently H, OH, CH3, OCH3 or OCH2CH3. More preferably for formula I: R5 is CH3; R1 and R4 are H; and R2 and R3, independently are H, OH, CH3, OCH3 or OCH2CH3. Even more preferably for formula I: R5 is CH3; R1, R2 and R4 are H; and R3 is OH. Preferably for formula II: n is 1 and R5 is OH; and R1, R2 and R3, independently are H, OH, CH3, OCH3 or OCH2CH3. More preferably for formula II: n is 1 and R5 is OH; R1 is H; and R2 and R3, independently are H, OH, CH3, OCH3 or OCH2CH3. Even more preferably for formula II: n is 1 and R5 is OH; and R1, R2 and R3 are H.
The present invention further provides a pharmaceutical composition comprising a quinone-related compound, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluant, wherein the compound comprises a compound of formula I or II wherein formula I is: 
wherein R1, R2, R3, and R4 are independently H, CH3, CH2CH3, OH, OCH3, OCH2CH3, or halo (Br, Cl, F, or I) and R5 is independently H, CH3, CH2CH3, OH, OCH3, OCH2CH3, or NR6R7 wherein R6 and R7 are independently H or C1-4alkyl; and wherein formula II is: 
wherein R1, R2 and R3 are independently as defined above and R8 is (CHxe2x95x90CH),xe2x80x94COxe2x80x94R5 wherein n=0, 1, 2, or 3 and R5 is independently as defined above for formula I.
Preferably for formula I: R5 is CH3; and R1, R2, R3 and R4 are independently H, OH, CH3, OCH3 or OCH2CH3. More preferably for formula I: R5 is CH3; R1 and R4 are H; and R2 and R3, independently are H, OH, CH3, OCH3 or OCH2CH3. Even more preferably for formula I: R5 is CH3; R1, R2 and R4 are H; and R3 is OH. Preferably for formula II: n is 1 and R5 is OH; and R1, R2 and R3, independently are H, OH, CH3, OCH3 or OCH2CH3. More preferably for formula II: n is 1 and R5 is OH; R1 is H; and R2 and R3, independently are H, OH, CH3, OCH3 or OCH2CH3. Even more preferably for formula II: n is 1 and R5 is OH; and R1, R2 and R3 are H.
Preferably, the pharmaceutical composition further comprises a steroid, a glucocorticoid, anti-TNFxcex1 antibody, anti-IL-1 antibody, anti-IFN-xcex3 antibody, IL-1RA, IL-10 or combinations thereof.
The present invention also provides a pharmaceutical composition comprising a quinone-related compound, and a pharmaceutically acceptable carrier, wherein the quinone-related compound forms a stable covalent interaction with an amino acid residue of a MIF protein. Preferably, the compound is a substituted iminoquinone compound or a substituted orthoquinone compound. Preferably, the pharmaceutical composition further comprises a steroid, a glucocorticoid, anti-TNFxcex1 antibody, anti-IL-1 antibody, anti-IFN-xcex3 antibody, IL-1RA, IL-10 or combinations thereof.
The present invention provides a method for treating inflammatory disorders (including, but not limited to, arthritis, proliferative vascular disease, EAE, ARDS (acute respiratory distress syndrome), cytokine-mediated toxicity, sepsis, septic shock, psoriasis, interleukin-2 toxicity, asthma, MIF-mediated conditions, autoimmune disorders (including but not limited to, rheumatoid arthritis, insulin-dependent diabetes, multiple sclerosis, graft versus host disease, lupus syndromes), tumor growth or angiogenesis, or any condition characterized by local or systemic MIF release or synthesis, comprising administering an effective amount of a quinone-related compound, wherein the quinone-related compound forms a stable covalent interaction with an amino acid residue of a MIF protein. Preferably, the compound is a substituted iminoquinone compound or a substituted orthoquinone compound.